Assessment of coronary heart disease with carbon dioxide

ABSTRACT

There are provided methods for diagnosing coronary heart disease in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to reach a predetermined PaCO2 in the subject to induce hyperemia, monitoring vascular reactivity in the subject and diagnosing the presence or absence of coronary heart disease in the subject, wherein decreased vascular reactivity in the subject compared to a control subject is indicative of coronary heart disease. There are also provided methods for increasing sensitivity and specificity of BOLD MRI.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. application Ser. No.15/672,162 filed on Aug. 8, 2017, which is a Continuation of U.S.application Ser. No. 14/075,918 filed on Nov. 8, 2013, which is aContinuation-in-Part of U.S. application Ser. No. 14/115,860 filed onNov. 5, 2013, which is a 371 of International Application No.PCT/US2012/036813 filed on May 7, 2012, which claims benefit of priorityof U.S. Provisional Application No. 61/482,956 filed on May 5, 2011. Theentire contents of each of the aforementioned applications isincorporated herein by reference.

GOVERNMENT RIGHTS

The invention was made with government support under Grant No. HL091989awarded by the National Institutes of Health. The government has certainrights to the invention.

FIELD

The disclosure is directed to methods for detecting coronary heartdisease using carbon dioxide (CO₂) to induce hyperemia and monitorvascular reactivity.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art. Coronary artery disease (CAD) leadsto narrowing of the small blood vessels that supply blood and oxygen tothe heart. Typically, atherosclerosis is the cause of CAD. As thecoronary arteries narrow, blood flow to the heart can slow down or stop,causing, amongst other symptoms, chest pain (stable angina), shortnessof breath and/or myocardial infarction. Numerous tests help diagnoseCAD. Such tests include coronary angiography/arteriography, CTangiography, echocardiogram, electrocardiogram (ECG), electron-beamcomputed tomography (EBCT), magnetic resonance angiography, nuclear scanand exercise stress test. Functional assessment of the myocardium (forexample the assessment of myocardium's oxygen status) requires that apatient's heart is stressed either via controlled exercise orpharmacologically.

Assessment of vascular reactivity in the heart is the hallmark of stresstesting in cardiac imaging aimed at understanding ischemic heartdisease. This is routinely done in Nuclear Medicine with radionuclideinjection (such as Thallium) in conjunction with exercise to identifyterritories of the heart muscle that are subtended by a suspectednarrowed coronary artery. In patients who are contraindicated forexercise stress-testing, this approach is typically used in conjunctionwith hyperemia inducing drugs, for example via adenosine infusion.Reduced coronary narrowing is expected to reduce hyperemic response andthe perfusion reserve. Since nuclear methods are hampered by the needfor radioactive tracers combined with limited imaging resolution, otherimaging methods, such as ultrasound (using adenosine along withmicrobubble contrast) and MRI (also using adenosine and variousconjugates of gadolinium (Gd) (first-pass perfusion) or alterations inoxygen saturation in response to hyperemia, also known as theBlood-Oxygen-Level-Dependent (BOLD) effect) are under clinicalinvestigation. Nonetheless, in patients who are contraindicated forexercise stress-testing, currently all imaging approaches requireadenosine to elicit hyperemia. However, adenosine has undesirable sideeffects (such as the feeling of “impending doom”, bradycardia,arrhythmia, transient or prolonged episode of asystole, ventricularfibrillation (rarely), chest pain, headache, dyspnea, and nausea),making it less than favorable for initial or follow-up studies and manypatients request that they do not undergo repeated adenosine stresstesting. Nonetheless repeated stress testing is indicated in asignificant patient population to assess the effectiveness ofinterventional or medical therapeutic regimens. In view of the sideeffects of hyperemia inducing drugs, there is a need for alternatives,which induce hyperemia in patients who are contraindicated for exercisestress-testing but do not cause the side effects caused by the existinghyperemia inducing drugs.

SUMMARY

There is provided herein the use of carbon dioxide to replacehyperemia-inducing drugs such as adenosine to induce hyperemia insubjects contra-indicated for exercise stress testing, so as to diagnosecoronary heart diseases without the undesirable side effects of drugssuch as adenosine. In an embodiment, the CO₂ levels are altered whilethe O₂ levels are held constant. In another embodiment, the CO₂ levelsare controlled by administering a blend of air and a controlled amountof a gas mixture comprising 20% oxygen and 80% carbon dioxide.

In an aspect, there are provided methods for diagnosing coronary heartdisease in a subject in need thereof comprising administering anadmixture comprising CO₂ to a subject to produce a hyperemic responsecorresponding to at least one selected increase in a subject's coronaryPaCO₂, monitoring vascular reactivity in the subject and diagnosing thepresence or absence of coronary heart disease in the subject. Thepresence of coronary disease can be detected by monitoring a parameterindicative of a disease-associated change in a vasoreactive response tothe at least one increase in PaCO₂ in at least one coronary blood vesselor region of the heart. The present technology is based, at least inpart, on the finding that such a change can be captured by monitoringthe quantum of change in a parameter affected by a change in PaCO₂, froman first PaCO₂ level to a second PaCO₂ level, for example a parametercorrelated with vasodilation such as increased blood flow.

An observation of a change in a vasodilatory response can be extended tocomparing responses among different subjects, wherein a decreasedvascular reactivity in a subject in need of a diagnosis compared to thatof a control subject is indicative of coronary heart disease. Thus,according to one embodiment, there is provided a method for assessinghyperemic response in a subject in need thereof comprising administeringan admixture comprising CO₂ to a subject to reach a predetermined PaCO₂in the subject to induce hyperemia, monitoring vascular reactivity inthe subject and assessing hyperemic response in the subject, whereindecreased vascular reactivity in the subject compared to a controlsubject is indicative of poor hyperemic response, thereby assessinghyperemic response in the subject in need thereof.

In some embodiments, methods provided herein are directed to assessingorgan perfusion in a subject in need thereof.

In some embodiments, methods provided herein are directed to assessingvascular reactivity of an organ in a subject in need thereof.

In another aspect, there are provided methods of producing coronaryvasodilation in a subject in need thereof comprising administering anadmixture comprising CO₂ to a subject to reach a predetermined PaCO₂ inthe subject so as to produce coronary vasodilation, thereby producingcoronary vasodilation in the subject.

In yet another aspect, there are provided methods for increasingsensitivity and specificity for BOLD MRI. The method includesadministering an admixture comprising CO₂ to a subject to reach apredetermined PaCO₂ in the subject to induce hyperemia and imaging themyocardium using MRI to assess a hypermic response in response to apredetermined modulation in PaCO₂. In some embodiments, imaging themyocardium comprises (i) obtaining free-breathing cardiac phase resolved3D myocardial BOLD images; (ii) registering and segmenting the images toobtain the myocardial dynamic volume; and (iii) identifying ischemicterritory and quantifying image volume.

In a further aspect, there is provided the use of a CO₂ containing gasfor inducing hyperemia in a subject in need of a diagnostic assessmentof coronary heart disease, wherein the CO₂ containing gas is used toattain at least one increase in a subject's coronary PaCO₂ sufficientfor diagnosing coronary heart disease from imaging data, wherein theimaging data is indicative of a cardiovascular-disease-associatedvasoreactive response to the least one increase in PaCO₂ in at least onecoronary blood vessel or region of the heart.

In another aspect, there is provided a method for inducing hyperemia ina subject in need of a diagnostic assessment of coronary heart diseasecomprising administering a CO₂ containing gas, attaining at least oneincrease in a subject's coronary PaCO₂ sufficient for diagnosingcoronary heart disease from imaging data and imaging the heart during aperiod in which the increase in PaCO₂ is measurable, wherein the imagingdata is indicative of a cardiovascular disease-associated vasoreactiveresponse in at least one coronary blood vessel or region of the heart.

In some embodiments, the at least one increase in the subject's PaCO₂ isselected to produce a coronary vasoreactive response sufficient forreplacing a hyperemia inducing drug in assessing coronary disease. Inone embodiment, the hyperemia inducing drug that is replaced isadenosine.

In some embodiments, the methods provided herein comprise attaining aparticular predetermined PaCO₂.

In some embodiments, the pre-determined PaCO₂ is patient specific, i.e.determined relative to a baseline steady level in the patient (alsoreferred to herein as a reference steady state). For example, thepre-determined PaCO₂ may be an 8 to 20 mm Hg increase relative to abaseline steady level measured at the time of testing in the patient. Inan embodiment, the pre-determined PaCO₂ is an increase of about 25 mm Hgrelative to a baseline steady level measured at the time of testing inthe patient. In some embodiments, the pre-determined PaCO₂ is anincrease of about 22 mm Hg to about 28 mm Hg, about 22 mm Hg, about 23mm Hg, about 24 mm Hg, about 25 mm Hg, about 26 mm Hg, about 27 mm Hg,or about 28 mm Hg relative to a baseline steady level measured at thetime of testing in the patient.

In some embodiments, the methods provided herein comprise administeringcarbon dioxide in a stepwise manner.

In some embodiments, the methods provided herein comprise administeringcarbon dioxide in a block manner.

In some embodiments, the CO₂ is administered via inhalation.

In some embodiments, the disease-associated coronary vasoreactiveresponse is assessed relative to a control subject.

In some embodiments, the PaCO₂ is increased and decreased repeatedly inthe subject.

In some embodiments, the at least one PaCO₂ produces at least an 8%-12%increase in a BOLD signal intensity.

In some embodiments, the disease-associated vasoreactive response is acompromised increase in blood flow.

In some embodiments, the imaging data is indicative of the presence orabsence of a two-fold increase in blood flow in a coronary artery.

In some embodiments the imaging data are obtained by MRI and the imagingmethod obtains input of a change in signal intensity of a BOLD MRIsignal.

In some embodiments, the imaging method is PET or SPECT and the measureof a disease-associated vasoreactive response is the presence or absenceof a threshold increase in blood flow.

In some embodiments, the at least one increase in PaCO₂ produces atleast a 10% increase in intensity of a BOLD MRI signal.

In some embodiments, the at least one increase in PaCO₂ produces a10-20% increase in intensity of a BOLD MRI signal.

In some embodiments, the methods provided herein comprise: (i) imagingthe myocardium to obtain free-breathing cardiac phase resolved 3Dmyocardial BOLD images; (ii) registering and segmenting the images toobtain the myocardial dynamic volume; and (iii) identifying ischemicterritory and quantifing image volume.

In some embodiments, the at least one PaCO₂ is at least a 10 mm Hgincrease from a first level which is determined to be between 30 and 55mm Hg. Optionally, the first level is first determined to be between 35and 45 mm Hg. In some embodiments, the at least one PaCO₂ is about a 25mm Hg increase from a first level which is determined to be betweenabout 20 and about 55 mm Hg. In some embodiments, the at least one PaCO₂is about a 22 mm Hg to about 28 mm Hg, about a 22 mm Hg, about a 23 mmHg, about a 24 mm Hg, about a 25 mm Hg, about a 26 mm Hg, about a 27 mmHg, or about a 28 mm Hg increase from a first level which is determinedto be between about 20 and about 55 mm Hg, e.g., about 20, about 30,about 35, about 40, about 45, about 50, or about 55 mm Hg. In oneembodiment, the at least one PaCO2 is about a 25 mm Hg increase from afirst level which is determined to be about 35 mm Hg.

In some embodiments, the sufficiency of the increase in PaCO₂ isdetermined by increasing PaCO₂ in a stepwise manner.

In some embodiments, the vasoreactive response is sufficient forobtaining a disease-associated change in BOLD MRI signal obtained byadministering CO₂ in a manner effective to alternate between two or morePaCO₂ levels over a period of time and using repeated BOLD MRImeasurements to statistically assess the hyperemic response.

In some embodiments, the coronary vasoreactive response corresponds to avasodilatory response produced by administering a hyperemia inducingdrug for a duration and in amount per unit of time effective to assesscoronary disease.

In some embodiments, the hyperemia inducing drug is adenosine, whereinadenosine is administered in a regimen of 140 milligrams/litre perminute over 4 to 6 minutes.

In some embodiments, the methods provided herein comprise admixing airwith a selected amount of a CO₂ containing gas controlled to obtain apredetermined size increase in PaCO₂ from a previous value, for examplea measured baseline value.

The CO₂ containing gas may contain, for example, 75 to 100% CO₂. In someembodiments the CO₂ containing gas comprises a percentage composition ofoxygen in the 18-23% range, optionally about 20%.

In another aspect, there is provided a method for diagnosing coronaryheart disease in a subject in need thereof comprising:

-   -   (i) administering an admixture comprising CO₂ to a subject in a        stepwise or block manner to reach a predetermined PaCO₂ in the        subject to induce hyperemia;    -   (ii) monitoring vascular reactivity in the subject; and    -   (iii) diagnosing the presence or absence of coronary heart        disease in the subject, wherein decreased vascular reactivity in        the subject compared to a control subject is indicative of        coronary heart disease,

thereby diagnosing coronary heart disease in the subject in needthereof.

As elaborated below, administering carbon dioxide to alter PaCO₂ inblock manner, is optionally repeated over time. Optionally carbondioxide is administered so as to alternate between two or more levels ofPaCO₂ over a period of time.

Vascular reactivity may be monitored using any one or more of a varietyof advanced imaging methods including without limitation positronemission tomography (PET), single photon emission computedtomography/computed tomography (SPECT), computed tomography (CT), andmagnetic resonance imaging (MRI), and the like. In some embodiments,vascular reactivity may be measured using FFR.

In one embodiment, an admixture of CO₂ and O₂ for inducing hyperemia isan admixture in which O₂ is present in the range of 19-22%, for exampleabout 20%. Such an admixture can be used, for example, for blending aCO₂ containing gas with air for inhalation In this embodiment, CO₂ maymake up the rest of the admixture (81-78% respectively) or there may bea third gas in the admixture.

In a further aspect, there are provided methods for deliveringcontrolled amounts of carbon dioxide for inspiration by a subject duringfree breathing in a cardiac imaging procedure, so as to attain at leastone altered level of carbon dioxide in the subject's arterial blood, theat least one altered level of carbon dioxide selected to induce aselected hyperemic response in the subject's myocardium over a timeperiod selected for imaging the hyperemic response, the hyperemicresponse predetermined to enable at least one segment of the subject'smyocardium with a relatively reduced hyperemic response to be identifiedin the cardiac imaging procedure.

In some embodiments, the methods comprise use of a gas flow controllerconfigured to deliver controlled amounts of carbon dioxide forinspiration by the subject during free breathing in a cardiac imagingprocedure, so as to attain the desired at least one altered level ofcarbon dioxide in the subject's arterial blood.

In some embodiments, the methods comprise use of a gas flow controllerconfigured to deliver controlled amounts of carbon dioxide forinspiration by the subject during free breathing in a cardiac imagingprocedure, so as to attain the selected hyperemic response in thesubject's myocardium. The selected hyperemic response may be, forexample, a two-fold increase in the subject's myocardial blood flowrelative to a measured baseline value in the subject, or a hyperemicresponse substantially the same as that obtained using ahyperemia-inducing drug such as adenosine (a reference standardhyperemic response), as described further herein.

In some embodiments, there are provided methods of controlling a gasflow controller wherein the gas flow controller is configured to delivercontrolled amounts of carbon dioxide for inspiration by a subject duringfree breathing in a cardiac imaging procedure, so as to attain theselected at least one altered level of carbon dioxide in the subject'sarterial blood and/or the selected hyperemic response in the subject'smyocardium.

In one embodiment, the disclosure is directed to a method for measuringa reduced hyperemic response in an ischemic territory of a subject'smyocardium during a cardiac imaging procedure, comprising deliveringcontrolled amounts of carbon dioxide for inspiration by a subject duringfree breathing so as to attain the selected at least one altered levelof carbon dioxide in the subject's arterial blood.

In one embodiment, the levels of oxygen in the subject's arterial bloodare maintained constant or substantially constant during the imagingprocedure.

In one embodiment, normoxia (i.e., normal levels of oxygen) ismaintained during the imaging procedure.

In one embodiment, the gas flow controller is configured to attain atleast one target end tidal partial pressure (PETCO₂) of carbon dioxidethat corresponds to the selected at least one altered level of carbondioxide.

In one embodiment, the gas flow controller is configured toindependently attain a target end tidal partial pressure of oxygen thatis constant, for example, at a baseline level for the subject at rest.

In one embodiment, the gas flow controller is configured to attain atleast one increase in the subject's coronary PaCO₂ sufficient forcardiac imaging, cardiac stress testing, diagnosing coronary heartdisease from imaging data, etc.

In one embodiment, the gas flow controller is configured to attain theselected at least one altered level of carbon dioxide in a block manner.

In one embodiment, the reduced hyperemic response is less than theselected hyperemic response, wherein the difference between the reducedhyperemic response and the selected hyperemic response is statisticallysignificant.

In one embodiment, the selected hyperemic response is an increase in thesubject's myocardial blood flow that is substantially the same as thatattained by administering a controlled amount of a hyperemia-inducingdrug, e.g., adenosine, over a pre-determined time period.

As used herein, two values are “substantially the same” if they areessentially the same within the error of measurement, if there is nostatistically significant difference between them, or if the differencebetween them is not “functionally significant”, i.e., does not affectoutcome in the methods provided herein. “Statistically significant”generally means that the difference between two values has a p-value of<0.05, i.e., has a 95% chance of representing a meaningful differencebetween the two values.

In some embodiments, the selected hyperemic response is characterizedwith reference to the manifestation of a stress perfusion defect in asubject with ischemic heart disease. For example, in some embodiments,the selected hyperemic response represents a myocardial stress perfusiondefect in segments of the heart that are substantially the same as thoseproduced by a controlled amount of a hyperemia-inducing drug such asadenosine. In some embodiments, the selected hyperemic responserepresents a total stress perfusion defect that is substantially thesame (e.g., statistically indistinguishable and/or functionallyindistinguishable in the methods described herein) from that produced bya controlled amount of a hyperemia-inducing drug such as adenosine. Forexample, the total reduction in myocardial perfusion volume as afraction of total LV volume (TRP, % LV) is substantially the same asthat produced by the hyperemia-inducing drug. In some embodiments, whenvisual scoring of a stress perfusion defect is evaluated, theconcordance between the total myocardial segments identified as truepositives and true negatives for presence of the perfusion defect aresubstantially the same as that determined using a hyperemia-inducingdrug such as adenosine. The hyperemic response induced in a subject by acontrolled amount (e.g., a clinical amount) of a hyperemia-inducing drugsuch as adenosine is also referred to herein as a “reference standardhyperemic response”.

In one embodiment, the hyperemia inducing drug is adenosine or an analogthereof. However, the hyperemia inducing drug is not meant to be limitedand other hyperemia inducing drugs may be used in methods providedherein.

In one embodiment, the selected hyperemic response is a two-foldincrease in the subject's myocardial blood flow relative to a measuredbaseline value in the subject.

In one embodiment, the selected hyperemic response is induced byattaining a carbon dioxide level of about 60 to about 65 mm of Hg for atleast about one to two minutes in the subject.

In one embodiment, the at least one altered level of carbon dioxide is a25 mm of Hg increase from a baseline level of carbon dioxide in thesubject. A “baseline level” of mmHg in the subject is the level in thesubject before beginning the procedure, i.e., before the delivery ofcarbon dioxide in accordance with the methods described herein. Forexample, the baseline level may be the level in the subject at rest(normocapnic level); the level in the subject when the subject isbreathing at a regulated elevated minute volume (hypocapnic level); orany predetermined or selected starting level for the subject beforeinitiating delivery of carbon dioxide. In some embodiments, for example,the baseline level may be lower than the level in the subject at rest(normal rest levels vary but are typically about 35 to about 45 mm Hg).

For example, if the subject's ventilation (minute volume) is regulatedto be faster than at rest, then the measured baseline level may be lowerthan normal rest level for the subject (a hypocapnic level). In oneembodiment, the baseline level is about 30 mm Hg for a subject whosePCO₂ at rest is about 40 mm Hg. In one embodiment, the baseline level isthe normocapnic level for the subject. In another embodiment, thebaseline level is the hypnocapnic level for the subject. In someembodiments, the baseline level is about 30 mm of Hg. In someembodiments, the baseline level is about 30 mm of Hg. It should beunderstood that the baseline level is not meant to be particularlylimited and will be selected by the skilled artisan.

In some embodiments, the at least one altered level of carbon dioxide isselected to provide a hyperemic response which is a two-fold increase inthe subject's myocardial blood flow relative to a measured baselinevalue in the subject. In some embodiments, the at least one alteredlevel of carbon dioxide is selected to provide a hyperemic responsewhich is substantially the same as that obtained using ahyperemia-inducing drug such as adenosine. In such embodiments, anyaltered level of carbon dioxide that provides the desired hyperemicresponse may be selected.

In some embodiments, the at least one altered level of carbon dioxide ismaintained stably in the patient for a time sufficient to induce andenable measurement of a level-related hyperemic response as describedherein, e.g., for a predetermined time.

In one embodiment, the cardiac imaging procedure is PET, optionally ¹³Nammonia PET. However, the cardiac imaging procedure is not meant to beparticularly limited. Any suitable imaging procedure known in the artmay be used with methods described herein.

The at least one altered level of carbon dioxide is generally a levelthat is maintained within a predetermined confined range so as to induceand enable measurement of a level-related hyperemic response. In thismanner, a dose-related response can be measured in a manner comparableto use of a hyperemia inducing drug. For example, a range of 1 to 3 mmof Hg can be maintained using known control algorithms for attainingtarget end tidal concentrations of carbon dioxide, for example, on abreath by breath basis. In some embodiments, the at least one alteredlevel of carbon dioxide is selected to provide a hyperemic responsewhich is substantially the same as the hyperemic response obtained by ahyperemia inducing drug such as adenosine (e.g., by a standard dose ofadenosine). In some embodiments, the at least one altered level ofcarbon dioxide is selected to provide a hyperemic response which is atwo-fold increase in the subject's myocardial blood flow relative to ameasured baseline value in the subject. In some embodiments, the atleast one altered level of carbon dioxide is selected to provide ahyperemic response which is an increase in the subject's myocardialblood flow relative to a measured baseline value in the subject, whichmay be substantially the same amount of increase in the subject'smyocardial blood flow as is obtained by a hyperemia inducing drug suchas adenosine, e.g., a two-fold or more than two-fold increase inmyocardial blood flow. In this context, the measured baseline value inthe subject is the myocardial blood flow measured in the subject beforebeginning the procedure, i.e., before the delivery of carbon dioxide inaccordance with the methods described herein.

In some embodiments, there are provided methods for cardiac imaging,cardiac stress testing, and the like in accordance with standardprocedures known in the art, wherein the use of a hyperemia inducingdrug in such procedures is replaced by delivery of controlled amounts ofcarbon dioxide for inspiration by a subject during free breathing, suchthat at least one altered level of carbon dioxide in the subject'sarterial blood is attained and/or at least a selected hyperemic responsein the subject's myocardial blood flow is induced. In some suchembodiments, the at least one altered level of carbon dioxide and/or theselected hyperemic response is substantially the same as that obtainedby use of the hyperemia inducing drug which is replaced by delivery ofCO₂ in the procedure. In some such embodiments, there are providedmethods for cardiac imaging, cardiac stress testing, measuring ahyperemic response, and the like in subjects in need thereof who haveconsumed caffeine or a caffeine-containing substance (e.g., chocolate,tea, coffee, etc.) prior to initiating the procedures described herein.Notably, unlike CO₂ some hyperemia-inducing drugs do not producereliable assessments in subjects who have consumed caffeine beforehand.

Methods and systems for controlling a gas delivery device during acardiac imaging procedure and/or for measuring a hyperemic response arealso provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

For a better understanding of the present invention, as well as otheraspects and further features thereof, reference is made to the followingdescription which is to be used in conjunction with the accompanyingdrawings, where:

FIG. 1 depicts, in accordance with an embodiment of the presentinvention, the vascular reactivity in dogs as measured by theBOLD-effect using medical-grade Carbogen (5% CO₂ and 95% O₂) with andwithout coronary artery stenosis.

FIG. 2 depicts myocardial BOLD MRI with CO₂ in canines under normocarbicand hypercarbic conditions under free breathing conditions.

FIG. 3 depicts myocardial BOLD response to step-wise PaCO₂ ramp up incanines while holding basal PaO₂ constant.

FIG. 4 depicts myocardial BOLD response to repeated (block)administration CO₂ response.

FIG. 5 depicts the Doppler flow through the left anterior descendingartery in response to PaCO₂ modulation while PaO₂ is held constant.

FIG. 6 depicts the Doppler flow through the LAD, RCA and LCX arteries inresponse to PaCO₂ modulation while PaO₂ is held constant.

FIG. 7 is a bar graph depicting the territorial myocardial BOLD responseto PaCO₂ modulations in canines while PaO₂ is held constant.

FIG. 8 is a bar graph depicting the BOLD effect associated with PaCO₂modulation in blood, muscle and air while PaO₂ is held constant.

FIG. 9 is a table summarizing the statistical BOLD data associated withthe PaCO₂ modulation in myocardial territories, blood, muscle and air,while PaO₂ is held constant.

FIG. 10 is a comparison of BOLD response to adenosine and PaCO₂ (whilePaO₂ is held constant).

FIG. 11 depicts the early findings of BOLD response to PaCO₂ in humans,while PaO₂ is held constant.

FIG. 12(a) depicts a simulated BOLD signal for a change in PaCO₂ (redline) with definitions for noise variability (σ=20) and response. FIG.12(b) depicts a relation between BOLD response (y-axis) and the numberof measurements (x-axis) required to establish statistical significance(color-coded p-values). For a given BOLD response, the number ofrepeated measurements (N) required for reliable assessment (p<0.05) of achange from baseline condition lies at the right of the white dottedline. For example, to reliably detect a BOLD response from a voxel withpeak BOLD signal response of 10%, greater than 8 measurements areneeded. The bar on the right gives the scale for p values associatedwith the statistical significance.

FIG. 13 is a table summarizing estimates of mean arterial CO2, O2, andhemodynamic variables of interest in group stenosis. SBP: SystolicArterial Blood Pressure; HR: Heart Rate; RPP: Rate Pressure Product(MAP×HR). *denotes P<0.05 in comparison to rest values.

FIG. 14 depicts global and regional myocardial blood flow response tohypercapnia and adenosine in intact canines. Panels A and B show thecorresponding dynamic radiotracer uptake curves, which show theincreased myocardial uptake responses to hypercapnia and adenosinestresses relative to rest. Panels C and D show the global mean MBF andthe corresponding MPR at rest and under hypercapnia and adenosine. *denotes P<0.05.

FIG. 15 depicts regional myocardial blood flow response to hypercapniaand adenosine in the presence of coronary stenosis. Panel A showsrepresentative short and long-axis PET images of peak myocardial uptakeof ¹³N-ammonia during hypercapnia of PaCO₂˜60 mmHg (CO₂), standardclinical dose of adenosine (Adenosine) and at rest with PaCO₂˜35 mmHg(Rest) in a canine with a LAD stenosis. Note the lower uptake of theradiotracer in the anterior lateral wall (lower signal in distal LADsegments, yellow arrows) under hypercapnia and adenosine. For the casein panel A, rest and stress MBF (under hypercapnia and adenosine) andcorresponding MPR are shown as polar maps in panel B. These images showmarked reduction in MBF and MPR in the LAD territory, which are visuallyevident and spatially concordant under hypercapnia and adenosine.

FIG. 16 depicts quantitative measurements of regional myocardial bloodflow response to hypercapnia and adenosine in the presence of coronarystenosis. Panels A and B show mean regional MBF at rest, hypercapnia andadenosine. Regional MBF under hypercapnia and adenosine showed goodcorrelation and agreement. Panels C and D show corresponding MPR underhypercapnia and adenosine with similar results. * denotes P<0.05compared to conditions of rest; and +denotes P<0.05 compared to LADunder stress.

FIG. 17 depicts total myocardial perfusion defect due to coronarystenosis under hypercapnia and adenosine. Panel A shows the perfusiondefects detected from the Change Analysis estimated from time-averagedmyocardial uptake images at rest and stress (hypercapnia and adenosine),the polar images highlighting total perfusion defects (right). Note thenear identical correspondence in the perfusion defect territoriesidentified in the slices and the whole heart under hypercapnia andadenosine. Panel B shows the mean TRP (% LV) under hypercapnia andadenosine. No significant difference in TRP (% LV) was observed underhypercapnia and adenosine. Panels C and D show results from linearregression and Bland-Altman analyses.

FIG. 18 depicts visual scoring of perfusion defects under hypercapniaand adenosine in the presence of LAD coronary stenosis. Visual scoring(counts) from segments in the stenosis studies are presented in a 3D barplot. Excellent correspondence in visual scoring between hypercapnia andadenosine is observable (high count rates along the diagonal).

FIG. 19 depicts global and regional myocardial blood flow response tohypercapnia and adenosine following caffeine administration. Panel Ashows representative short- and long-axis PET images acquired duringpeak myocardial uptake of ¹³N-ammonia under hypercapnia of PaCO₂˜60 mmHg(CO₂), standard adenosine dose (Adenosine) and at baseline conditionswith PaCO₂˜35 mmHg (Rest) post caffeine administration. These visualresults show that the increase in myocardial uptake of radiotracerrelative to rest to occur only under hypercapnia; but not underadenosine. For the case in panel A, rest and stress MBF (underhypercapnia and adenosine) and corresponding MPR are shown as polar mapsin panel B. Panel C shows the MBF at rest before (Caff(−)) and after(Caff(+)) caffeine administration.

FIG. 20 depicts MBF and MPR response under hypercapnia and adenosinefollowing caffeine administration. Panel A shows the global and regionalmean MBF at rest and under hypercapnia and adenosine following caffeineinfusion (Caff+). Panel C shows the results from linear regressionanalysis between regional MBF under adenosine and hypercapnia. Panels Band D show corresponding MPR response. * denotes P<0.05.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3^(rd) ed., J. Wiley & Sons (New York, N.Y. 2001); March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^(th)ed., J. Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel,Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring HarborLaboratory Press (Cold Spring Harbor, N.Y. 2001), provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of the disease condition,preventing the disease condition from worsening, curing the diseasecondition, preventing the disease condition from developing, loweringthe chances of a patient developing the disease condition and prolonginga patient's life or life expectancy.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition,prevent the pathologic condition, pursue or obtain beneficial results,or lower the chances of the individual developing the condition even ifthe treatment is ultimately unsuccessful. Those in need of treatmentinclude those already with the condition as well as those prone to havethe condition or those in whom the condition is to be prevented.

“Carbogen” as used herein is an admixture of carbon dioxide and oxygen.The amounts of carbon dioxide and oxygen in the admixture may bedetermined by one skilled in the art.

Medical grade carbogen is typically 5% CO₂ and 95% O₂. In various otherembodiments, carbon dioxide used to induce hyperemia may be an admixtureof ranges including but not limited to 94% O₂ and 6% CO₂, 93% O₂ and 7%CO₂, 92% O₂ and 8% CO₂, 91% O₂ and 9% CO₂, 90% O₂ and 10% CO₂, 85% O₂and 15% CO₂, 80% O₂ and 20% CO₂, 75% O₂ and 25% CO₂ and/or 70% O₂ and30% CO₂. Optionally, for blending with air, the CO₂ containing gascomprises 20% oxygen.

“BOLD” as used herein refers to blood-oxygen-level dependence.

The term “about” is used herein to indicate that a value includes aninherent variation of error for the device or the method being employedto determine the value.

A “vascular-disease-associated” coronary vasoreactive response means atype and/or quantum of vasoreactive response elicited by cardiac stresstesting (e.g. exercise or administration of a hyperemic drug or a CO₂containing gas) as demonstrable in an imaging study using one or morediagnostic imaging parameters of the type suitable to diagnose coronaryvascular disease. For example, with respect to PET and SPECT, a normalresponse would be considered a four to five-fold increase in blood flow.With respect to BOLD MRI imaging, a 10-12+% increase in BOLD signalwould be considered normal. Disease associated responses are those whichare not normal in varying significant degrees among which, as evidenceof disease, benchmarks may be adopted to categorize differences whichrepresent a clearer-cut diagnosis or a progression of disease thatwarrants greater follow-up or more proactive treatment, for example aless than two-fold increase in blood flow as measured by PET or SPECT(typically measured in ml. of blood/min/gm of tissue). Accordingly, abenchmark which represents a change from a value that cliniciansdescribed as “normal” which is at least statistically significant andoptionally is also comparable to a standard for cardiac stress testingadopted by clinicians with respect to inducing stress represents aclear-cut benchmark for using CO₂ as a vasoactive stress stimulus.

A targeted increase in PaCO₂ will be selected to cause a similarvasoreactive response in normal and diseased tissue. From the standpointof statistical significance, it will be appreciated that selection of adiscriminatory increase in PaCO₂ may depend on whether or not repeatmeasurements are made, for example, the number of repeat measurements ofa BOLD signal intensity that are made while at lower and increased PaCO₂levels.

Current methods for inducing hyperemia in subjects include the use ofcompounds such as adenosine, analogs thereof and/or functionalequivalents thereof. However, such compounds (for example, adenosine)have adverse side effects including bradycardia, arrhythmia, transientor prolonged episode of asystole, ventricular fibrillation (rarely),chest pain, headache, dyspnea, and nausea, making it less than favorablefor initial or follow-up studies.

The technology described herein is directed to the use of CO₂ instead ofhyperemia-inducing drugs, in view of their side effects, to assessmyocardial response and risk of coronary artery diseases. To date,however, it has not been possible to independently control arterial CO₂and O₂, hence direct association of the influence of partial pressure ofCO₂ (PaCO₂) on coronary vasodilation has been difficult to determine.With the development of gas flow controller devices designed to controlgas concentrations in the lungs and blood (for example, RespirACT™,Thornhill Research, WO/2013/0082703), it is now possible to preciselycontrol the arterial CO₂, while, in some embodiments, holding O₂constant. With such devices, the desired PaCO₂ changes are rapid (1-2breaths) and are independent of minute ventilation. The inventors areamong the first adopters of such devices for the assessment ofmyocardial response to CO₂.

The methods provided herein are believed to be the first to usemodulation of CO₂ levels to show that the carbon dioxide can have thesame effect as the clinical dose of other hyperemia-inducing drugs suchas adenosine but without the side effects. We report herein thathyperemia is induced by administering an admixture comprising apredetermined amount of CO₂ to a subject in need thereof to assessmyocardial response, evaluate coronary artery disease and identifyischemic heart disease. In an embodiment, hyperemia is induced byindependently altering the administered CO₂ level while holding oxygen(O₂) constant to assess myocardial response, evaluate coronary arterydisease and identify ischemic heart disease. A subject's myocardialresponse after administration of CO₂ may be monitored using variousimaging techniques such as MRI.

Cardiac Stress Testing

When exercise stress testing is contra-indicated (in nearly 50% ofpatients), existing imaging modalities use adenosine (or its analoguessuch as dipyridamole or regadenoson) to induce hyperemia. However, asdescribed above, adenosine or analogs thereof or functional equivalentsthereof, are well known for their adverse side effects such asbradycardia, arrhythmia, transient or prolonged episode of asystole,ventricular fibrillation (rarely), chest pain, headache, dyspnea, andnausea, making it less than favorable for initial or follow-up studies.Direct measures of ischemic burden may be determined on the basis ofsingle-photon emission computed tomography (SPECT/SPET), positronemission tomography (PET), myocardial contrast echocardiography (MCE),and first-pass perfusion magnetic resonance imaging (FPP-MRI). SPECT andPET use radiotracers as contrast agents. While SPECT and PET studiesaccount for approximately 90% of myocardial ischemia-testing studies,the sensitivity and specificity for both methods combined for thedetermination of severe ischemia is below 70%. Both MCE and FPP-MRI arerelatively newer approaches that require the use of exogenous contrastmedia and intravenous pharmacological stress agent (such as adenosine),both carrying significant risks and side effects in certain patientpopulations.

BOLD-MRI

An alternate method, BOLD (Blood-Oxygen-Level-Dependent) MRI, relies onendogenous contrast mechanisms (changes in blood oxygen saturation, %O₂) to identify ischemic territories. The potential benefits of BOLD MRIfor detecting global or regional myocardial ischemia due to coronaryartery disease (CAD) were demonstrated at least a decade ago. Although anumber of pilot clinical studies have demonstrated the feasibility ofusing BOLD MRI for identifying clinically significant myocardialischemia due to CAD, the method is inherently limited by sensitivity andspecificity due to low BOLD contrast-to-noise ratio (CNR). In someembodiments of methods provided herein, the repeatability of BOLD MRIusing CO₂ provides the means to improve sensitivity and specificity,which is not possible using adenosine or analogs thereof.

In some embodiments, there is provided a method for increasing thesensitivity and specificity of BOLD MRI. The method includesadministering an admixture comprising CO₂ to a subject in need thereofto induce hyperemia and imaging the myocardium using MRI to assess ahypermic response in response to a predetermined modulation in PaCO₂.Insome embodiments, this method utilizes (i) an individualized targetedchange in arterial partial pressure of CO₂ (PaCO₂) as the non-invasivevasoactive stimulus, (ii) fast, high-resolution, 4D BOLD MRI at 3T and(iii) statistical models (for example, the generalized linear model(GLM) theory) to derive statistical parametric maps (SPM) to reliablydetect and quantify the prognostically significant ischemic burdenthrough repeated measurements (i.e., in a data-driven fashion). In someembodiments, the method for increasing the sensitivity and specificityof BOLD MRI comprises (i) obtaining free-breathing cardiacphase-resolved 3D myocardial BOLD images (under different PaCO₂ statesestablished via inhalation of an admixture of gases comprising of CO₂);(ii) registering and segmenting the images to obtain the myocardialdynamic volume; and (iii) identifying ischemic territory and quantifyingimage volume.

Obtaining the Images

The first step in increasing the sensitivity and specificity of BOLD MRIis to obtain free-breathing cardiac phase resolved 3D myocardial BOLDimages. Subjects are placed on the MRI scanner table, ECG leads areplaced, and necessary surface coils are positioned.

Subsequently the subjects' hearts are localized and the cardiac shimprotocol is prescribed over the whole heart. K-space lines, time stampedfor trigger time are collected using cine SSFP acquisition with imageacceleration along the long axis. Central k-space lines corresponding toeach cardiac phase will be used to derive the center of mass (COM)curves along the z-axis via 1-D fast Fourier transform (FFT). Based onthe COM curves, the k-space lines from each cardiac phase will be sortedinto 1-30 bins, each corresponding to a respiratory state with the firstbin being the reference bin (end-expiration) and the last bincorresponding to end inspiration.

To minimize the artifacts from under sampling, the data will beprocessed with a 3D filter, followed by re-gridding the k-space lines,application of a spatial mask (to restrict the registration to region ofthe heart) and performing FFT to obtain the under sampled 3D image foreach respiratory bin. Using the end-expiration image as the referenceimage, images from all bins (except bin 1) are registered using kitssuch as Insight Tool Kit (freely available from www.itk.org), or anequivalent software platform, in an iterative fashion and the transformparameters will be estimated for rotation, scaling, shearing, andtranslation of heart between the different respiratory bins. The k-spacedata will again be divided into 1 to 30 respiratory bins, re-gridded,transformed to the reference image (3D affine transform), summedtogether, and the final 3D image will be reconstructed. Imagingparameters may be TR=3.0 to 10 ms and flip angle=1° to 90°. In thisfashion, 3D cine data under controlled PaCO₂ values (hypo-andhyper-carbic states) are collected.

Registration and Segmentation of Images

The next step in increasing the sensitivity and specificity of BOLD MRIis registration and segmentation of the images to obtain the myocardialdynamic volume. The pipeline utilizes MATLAB and C++ using the ITKframework or an equivalent software platform. The myocardial MR imagesobtained with repeat CO₂ stimulation blocks will be loaded in MATLAB (oran equivalent image processing platform) and arranged in afour-dimensional (4D) matrix, where the first 3 dimensions representvolume (voxels) and the fourth dimension is time (cardiac phase).Subsequently, each volume is resampled to achieve isotropic voxel size.End-systole (ES) are identified for each stack based on our minimumcross-correlation approach. A 4D non-linear registration algorithm isused to find voxel-to-voxel correspondences (deformation fields) acrossall cardiac phases. Using the recovered deformation, all cardiac phasesare wrapped to the space of ES, such that all phases are aligned to ES.The next step is to recover the transformations across all ES imagesfrom repeat CO₂ blocks and bring them to the same space using adiffeomorphic volume registration tool, such as ANTs. Upon completion,all cardiac phases from all acquisitions will be spatially aligned tothe space of ES of the first acquisition (used as reference) and allphase-to-phase deformations and acquisition-to-acquisitiontransformations will be known. An expert delineation of the myocardiumin the ES of the first (reference) acquisition will then be performed.Based on the estimated deformation fields and transformations, thissegmentation is propagated to all phases and acquisitions, resulting infully registered and segmented myocardial dynamic volumes.

Image Analysis to Identify and Quantify Ischemic Territories

The final step needed for increasing the sensitivity and specificity ofBOLD MRI is identifying ischemic territory and quantifying image volume.Since BOLD responses are optimally observed in systolic frames, only Lsystolic cardiac volumes (centered at ES) are retained from each fullyregistered and segmented 4D BOLD MR image set obtained above. Only thosevoxels contained in the myocardium are retained and the correspondingRPP (rate-pressure-product) and PaCO₂ are noted. Assuming N acquisitionsper CO₂ state (hypocarbic or hypercarbic) and K, CO₂ stimulation blocks,and each cardiac volume consists of n x m x p (x=multiplication)isotropic voxels, build a concatenated fully registered 4D datasetconsisting of nxmxpxt pixels, where x=multiplication and t=L×K×N, andexport this dataset in NIFTI (or an equivalent) format using standardtools. The 4D dataset is loaded into a voxel-based statistical modelfitting (such as FSL-FEAT developed for fMRI), to fit the model for eachvoxel. The statistical analysis outputs a P-statistic volume, i.e., theSPM, where for each voxel in the myocardium the p-value of thesignificance of the correlation to the model is reported. Thestatistical parametric maps (SPM) are thresholded by identifying thevoxels that have p<0.05. Those voxels are identified as hyperemic forresponding to the CO₂ stimulation. The total number of hyperemic voxels(V_(H)) are counted and their relative volume (V_(RH)=V_(H)/total voxelsin myocardium) is determined. The voxels that do not respond to CO₂stimulation (on SPM) are identified as ischemic and used to generate abinary 3D map of ischemic voxels (3D-ISCH_(map)). In addition, totalischemic voxels (V_(I)) and the relative ischemic volume (V_(RI)=V_(I)/total myocardial voxels) are determined.

The above methods provide ischemic volumes that can be reliablyidentified on the basis of statistical analysis applied to repeatedlyacquire 4D BOLD images under precisely targeted changes in PaCO₂. Thesevolumes are closely related to the clinical index of fractional flowreserve FFR.

FFR

An additional method, fractional flow reserve (FFR) is used in coronarycatheterization to measure pressure differences across a coronary arterystenosis to determine the likelihood that the stenosis impedes oxygendelivery to the heart muscle (myocardial ischemia). Fractional flowreserve measures the pressure behind (distal to) a steno sis relative tothe pressure before the stenosis, using adenosine or papaverine toinduce hyperemia. A cut-off point of 0.75 to 0.80 has been used whereinhigher values indicate a non-significant stenosis and lower valuesindicate a significant lesion. FFR, determined as the relative pressuredifferences across the stenotic coronary artery has emerged as the newstandard for determining clinically significant ischemia (FFR≤0.75).However, it is invasive, expensive, and exposes the patient to ionizingradiation and the side-effects of the use of adenosine. In view of theside-effects of adenosine discussed above, Applicants propose usingcarbon dioxide instead of adenosine to induce hyperemia, byadministering to a subject an admixture comprising CO₂ to reach apredetermined PaCO₂ in the subject to induce hyperemia. In someembodiments, the admixture comprises any one or more of carbon dioxide,oxygen, and nitrogen, e.g., carbon dioxide, oxygen and nitrogen; carbondioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxidealone. In one embodiment, the amounts of CO₂ and O₂ administered areboth altered. In another embodiment, the amount of CO₂ administered isaltered to a predetermined level while the amount of O₂ administered isheld constant. In various embodiments, the amounts of any one or more ofCO₂, O₂ and N₂ in an admixture are changed or held constant as would bereadily apparent to a person having ordinary skill in the art.

Methods

There are provided herein methods for diagnosing coronary heart diseasein a subject in need thereof comprising administering an admixturecomprising CO₂ to a subject to reach a predetermined PaCO₂ in thesubject to induce hyperemia, monitoring vascular reactivity in thesubject and diagnosing the presence or absence of coronary heart diseasein the subject, wherein decreased vascular reactivity in the subjectcompared to a control subject is indicative of coronary heart disease.In an embodiment, CO₂ is administered via inhalation. In anotherembodiment, CO₂ levels are altered while the O₂ levels remain unchangedso that the PaCO₂ is changed independently of the O₂ level. In a furtherembodiment, vascular reactivity is monitored using imagining techniquesdeemed appropriate by one skilled in the art, including but not limitedto any one or more of positron emission tomography (PET), single photonemission computed tomography/computed tomography (SPECT), computedtomography (CT), magnetic resonance imaging (MRI), functional magneticresonance imaging (fMRI), single photon emission computedtomography/computed tomography (SPECT/CT), positron emissiontomography/computed tomography (PET/CT), ultrasound, electrocardiogram(ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO),and electron spin resonance (ESR), and/or any combination of the imagingmodalities such as (PET/MR), PET/CT, and/or SPECT/MR. In an embodiment,vascular reactivity is monitored using free-breathing BOLD MRI. In someembodiments, the admixture comprises any one or more of carbon dioxide,oxygen and nitrogen, e.g., carbon dioxide, oxygen and nitrogen; carbondioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxidealone. In one embodiment, the amounts of CO₂ and O₂ administered areboth altered. In another embodiment, the amount of CO₂ administered isaltered to a predetermined level while the amount of O₂ administered isheld constant. In various embodiments, the amounts of any one or more ofCO₂, O₂ and N₂ in an admixture are changed or held constant as would bereadily apparent to a person having ordinary skill in the art.

The invention also provides a method for assessing hyperemic response ina subject in need thereof comprising administering an admixturecomprising CO₂ to a subject to reach a predetermined PaCO₂ in thesubject to induce hyperemia, monitoring vascular reactivity in thesubject and assessing hyperemic response in the subject, whereindecreased vascular reactivity in the subject compared to a controlsubject is indicative of poor hyperemic response, thereby assessinghyperemic response in the subject in need thereof. This method may alsobe used to assess organ perfusion and assess vascular reactivity. In anembodiment, CO₂ is administered via inhalation. In another embodiment,CO₂ levels are altered while the O₂ levels remain unchanged so that thePaCO₂ is changed independently of the O₂ level. In a further embodiment,vascular reactivity is monitored using imagining techniques deemedappropriate by one skilled in the art, including but not limited to anyone or more of positron emission tomography (PET), single photonemission computed tomography/computed tomography (SPECT), computedtomography (CT), magnetic resonance imaging (MRI), functional magneticresonance imaging (fMRI), single photon emission computedtomography/computed tomography (SPECT/CT), positron emissiontomography/computed tomography (PET/CT), ultrasound, electrocardiogram(ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO),and electron spin resonance (ESR), and/or any combination of the imagingmodalities such as (PET/MR), PET/CT, and/or SPECT/MR. In an embodiment,vascular reactivity is monitored using free-breathing BOLD MRI. In someembodiments, the admixture comprises any one or more of carbon dioxide,oxygen and nitrogen, e.g., carbon dioxide, oxygen and nitrogen; carbondioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxidealone. In one embodiment, the amounts of CO₂ and O₂ administered areboth altered. In another embodiment, the amount of CO₂ administered isaltered to a predetermined level while the amount of O₂ administered isheld constant. In various embodiments, the amounts of any one or more ofCO₂, O₂ and N₂ in an admixture are changed or held constant as would bereadily apparent to a person having ordinary skill in the art.

The invention is further directed to methods for producing coronaryvasodilation in a subject in need thereof comprising providing acomposition comprising CO₂ and administering the composition comprisingCO₂ to a subject to reach a predetermined PaCO₂ in the subject so as toproduce coronary vasodilation in the subject, thereby producing coronaryvasodilation in the subject. In an embodiment, CO₂ is administered viainhalation. In another embodiment, CO₂ levels are altered while the O₂levels remain unchanged so that the PaCO₂ is changed independently ofthe O₂ level. In a further embodiment, vascular reactivity is monitoredusing imagining techniques deemed appropriate by one skilled in the art,including but not limited to any one or more of positron emissiontomography (PET), single photon emission computed tomography/computedtomography (SPECT), computed tomography (CT), magnetic resonance imaging(MRI), functional magnetic resonance imaging (fMRI), single photonemission computed tomography/computed tomography (SPECT/CT), positronemission tomography/computed tomography (PET/CT), ultrasound,electrocardiogram (ECG), Electron-beam computed tomography (EBCT),echocardiogram (ECHO), and electron spin resonance (ESR) and/or anycombination of the imaging modalities such as (PET/MR), PET/CT, and/orSPECT/MR. In an embodiment, vascular reactivity is monitored usingfree-breathing BOLD MRI. In some embodiments, the admixture comprisesany one or more of carbon dioxide, oxygen and nitrogen, e.g., carbondioxide, oxygen and nitrogen; carbon dioxide and oxygen; carbon dioxideand nitrogen; or carbon dioxide alone. In one embodiment, the amounts ofCO₂ and O₂ administered are both altered. In another embodiment, theamount of CO₂ administered is altered to a predetermined level while theamount of O₂ administered is held constant. In various embodiments, theamounts of any one or more of CO₂, O₂ and N₂ in an admixture are changedor held constant as would be readily apparent to a person havingordinary skill in the art.

The invention also provides a method for assessing tissue and/or organperfusion in a subject in need thereof comprising administering anadmixture comprising CO₂ to a subject to reach a predetermined PaCO₂ inthe subject to induce hyperemia, monitoring vascular reactivity in thetissue and/or organ and assessing tissue and/or organ perfusion byassessing the hyperemic response in the subject, wherein decreasedvascular reactivity in the subject compared to a control subject isindicative of poor hyperemic response and therefore poor tissue and/ororgan perfusion. In an embodiment, CO₂ is administered via inhalation.In another embodiment, CO₂ levels are altered while the O₂ levels remainunchanged so that the PaCO₂ is changed independently of the O₂ level. Ina further embodiment, vascular reactivity is monitored using imaginingtechniques deemed appropriate by one skilled in the art, including butnot limited to any one or more of positron emission tomography (PET),single photon emission computed tomography/computed tomography (SPECT),computed tomography (CT), magnetic resonance imaging (MRI), functionalmagnetic resonance imaging (fMRI), single photon emission computedtomography/computed tomography (SPECT/CT), positron emissiontomography/computed tomography (PET/CT), ultrasound, electrocardiogram(ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO),and electron spin resonance (ESR), and/or any combination of the imagingmodalities such as (PET/MR), PET/CT, and/or SPECT/MR. In an embodiment,vascular reactivity is monitored using free-breathing BOLD MRI. In someembodiments, the admixture comprises any one or more of carbon dioxide,oxygen and nitrogen, e.g., carbon dioxide, oxygen and nitrogen; carbondioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxidealone. In one embodiment, the amounts of CO₂ and O₂ administered areboth altered. In another embodiment, the amount of CO₂ administered isaltered to a predetermined level while the amount of O₂ administered isheld constant. In various embodiments, the amounts of any one or more ofCO₂, O₂ and N₂ in an admixture are changed or held constant as would bereadily apparent to a person having ordinary skill in the art.

In some embodiments, the admixture comprising CO₂ is administered athigh doses for short duration or at low doses for longer durations. Forexample, administering the admixture comprising CO₂ at high doses of CO₂for a short duration comprises administering any one or more of 40 mmHgto 45 mmHg, 45 mmHg to 50 mmHg, 50 mmHg to 55 mmHg, 55 mmHg CO₂ to 60 mmHg CO₂, 60 mmHg CO₂ to 65 mm Hg CO₂, 65 mmHg CO₂ to 70 mm Hg CO₂, 70mmHg CO₂ to 75 mm Hg CO₂, 75 mmHg CO₂ to 80 mm Hg CO₂, 80 mmHg CO₂ to 85mm Hg CO₂ or a combination thereof, for about 20 minutes, 15 minutes, 10minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4minutes, 3 minutes, 2 minutes, 1 minute or a combination thereof. Invarious embodiments, the predetermined levels of CO₂ are administered sothat the arterial level of CO₂ reaches the PaCO₂ of any one or more ofthe above ranges. In an embodiment, the predetermined levels of CO₂ areadministered so that the arterial level of CO₂ reaches the PaCO₂ ofabout 60 mm Hg.

For example, administering low doses of predetermined amounts of CO₂ fora longer duration comprises administering the predetermined amount ofCO₂ at any one or more of about 30 mmHg CO₂ to about 35 mmHg CO₂, about35 mmHg CO₂ to about 40 mmHg CO₂, about 40 mmHg CO₂ to about 45 mmHgCO₂, about 60 mmHg CO₂ to about 65 mmHg CO₂, or a combination thereoffor any one or more of about 20 to 24 hours, about 15 to 20 hours, about10 to 15 hours, about 5 to 10 hours, about 4 to 5 hours, about 3 to 4hours, about 2 to 3 hours, and about 1 to 2 hours, or a combinationthereof, before inducing hyperemia. In various embodiments, thepredetermined levels of CO₂ are administered so that the arterial levelof CO₂ reaches the PaCO₂ of any one or more of the above ranges.

In still further embodiments, the predetermined levels of CO₂ areadministered so that the arterial level of CO₂ reaches a PaCO₂ that isincreased by about 25 mmHg in a subject, i.e., an about 25 mm Hgincrease in PaCO₂ is achieved in the subject after the CO₂administration. In an embodiment, the predetermined levels of CO₂ areadministered so that the arterial level of CO₂ reaches the PaCO₂ ofabout 60 mm Hg. For example, PaCO₂ may be altered from a baseline levelof about 35 mm Hg to about 60 mm Hg in the subject. In some embodiments,the PaCO₂ is increased by about 22 mm Hg to about 28 mm Hg in thesubject, or by about 22 mm Hg, about 23 mm Hg, about 24 mm Hg, about 25mm Hg, about 26 mm Hg, about 27 mm Hg, or about 28 mm Hg in the subject.

In some embodiments, the predetermined levels of CO₂ are administered sothat the hyperemic response in the subject is an about two-fold increasein the subject's myocardial blood flow relative to a measured baselinevalue in the subject (e.g., value before administration of CO₂).

In some embodiments, the predetermined levels of CO₂ are administered sothat the hyperemic response in the subject is substantially the same asthe hyperemic response obtained using a hyperemia-inducing drug such asadenosine, e.g., is substantially the same as a reference standard. Insome embodiments, the predetermined levels of CO₂ are selected to inducea selected hyperemic response in the subject. In some embodiments, theselected hyperemic response is a reference standard hyperemic response.In some embodiments, the selected hyperemic response is a hyperemicresponse that is sufficient to show a response deficit in ischemictissue, i.e. sufficient to enable imaging of a reduced hyperemicresponse in ischemic tissue in at least one segment of the subject'smyocardium in a cardiac imaging procedure.

In one embodiment, CO₂ is administered in a stepwise manner. In anotherembodiment, administering carbon dioxide in a stepwise manner includesadministering carbon dioxide in 5 mmHg increments in the range of anyone or more of 10 mmHg to 100 mmHg CO₂, 20 mmHg to 100 mmHg CO₂, 30 mmHgto 100 mmHg CO₂, 40 mmHg to 100 mmHg CO₂, 50 mmHg to 100 mmHg CO₂, 60mmHg to 100 mmHg CO₂, 10 mmHg to 90 mmHg CO₂, 20 mmHg to 90 mmHg CO₂, 30mmHg to 90 mmHg CO₂, 40 mmHg to 90 mmHg CO₂, 50 mmHg to 90 mmHg CO₂, 60mmHg to 90 mmHg CO₂, 10 mmHg to 80 mmHg CO₂, 20 mmHg to 80 mmHg CO₂, 30mmHg to 80 mmHg CO₂, 40 mmHg to 80 mmHg CO₂, 50 mmHg to 80 mmHg CO₂, 60mmHg to 80 mmHg CO₂, 10 mmHg to 70 mmHg CO₂, 20 mmHg to 70 mmHg CO₂, 30mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHg to 70 mmHg CO₂, 60mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHg to 70 mmHg CO₂, 30mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHg to 70 mmHg CO₂, 60mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHg to 60 mmHg CO₂, 30mmHg to 60 mmHg CO₂, 40 mmHg to 60 mmHg CO₂ and 50 mmHg to 60 mmHg CO₂.In various embodiments, the predetermined levels of CO₂ are administeredso that the arterial level of CO₂ reaches the PaCO₂ of any one or moreof the above ranges.

In another embodiment, administering carbon dioxide in a stepwise mannerincludes administering carbon dioxide in 10 mmHg increments in the rangeof any one or more of 10 mmHg to 100 mmHg CO₂, 20 mmHg to 100 mmHg CO₂,30 mmHg to 100 mmHg CO₂, 40 mmHg to 100 mmHg CO₂, 50 mmHg to 100 mmHgCO₂, 60 mmHg to 100 mmHg CO₂, 10 mmHg to 90 mmHg CO₂, 20 mmHg to 90 mmHgCO₂, 30 mmHg to 90 mmHg CO₂, 40 mmHg to 90 mmHg CO₂, 50 mmHg to 90 mmHgCO₂, 60 mmHg to 90 mmHg CO₂, 10 mmHg to 80 mmHg CO₂, 20 mmHg to 80 mmHgCO₂, 30 mmHg to 80 mmHg CO₂, 40 mmHg to 80 mmHg CO₂, 50 mmHg to 80 mmHgCO₂, 60 mmHg to 80 mmHg CO₂, 10 mmHg to 70 mmHg CO₂, 20 mmHg to 70 mmHgCO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHg to 70 mmHgCO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHg to 70 mmHgCO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHg to 70 mmHgCO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHg to 60 mmHgCO₂, 30 mmHg to 60 mmHg CO₂, 40 mmHg to 60 mmHg CO₂ and 50 mmHg to 60mmHg CO₂. In various embodiments, the predetermined levels of CO₂ areadministered so that the arterial level of CO₂ reaches the PaCO₂ of anyone or more of the above ranges.

In a further embodiment, administering carbon dioxide in a stepwisemanner includes administering carbon dioxide in 20 mmHg increments inthe range of any one or more of 10 mmHg to 100 mmHg CO₂, 20 mmHg to 100mmHg CO₂, 30 mmHg to 100 mmHg CO₂, 40 mmHg to 100 mmHg CO₂, 50 mmHg to100 mmHg CO₂, 60 mmHg to 100 mmHg CO₂, 10 mmHg to 90 mmHg CO₂, 20 mmHgto 90 mmHg CO₂, 30 mmHg to 90 mmHg CO₂, 40 mmHg to 90 mmHg CO₂, 50 mmHgto 90 mmHg CO₂, 60 mmHg to 90 mmHg CO₂, 10 mmHg to 80 mmHg CO₂, 20 mmHgto 80 mmHg CO₂, 30 mmHg to 80 mmHg CO₂, 40 mmHg to 80 mmHg CO₂, 50 mmHgto 80 mmHg CO₂, 60 mmHg to 80 mmHg CO₂, 10 mmHg to 70 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 60 mmHg CO₂, 30 mmHg to 60 mmHg CO₂, 40 mmHg to 60 mmHg CO₂ and 50mmHg to 60 mmHg CO₂. In various embodiments, the predetermined levels ofCO₂ are administered so that the arterial level of CO₂ reaches the PaCO₂of any one or more of the above ranges.

In a further embodiment, administering carbon dioxide in a stepwisemanner includes administering carbon dioxide in 25mmHg increments in therange of any one or more of 10 mmHg to 100 mmHg CO₂, 20 mmHg to 100 mmHgCO₂, 30 mmHg to 100 mmHg CO₂, 40 mmHg to 100 mmHg CO₂, 50 mmHg to 100mmHg CO₂, 60 mmHg to 100 mmHg CO₂, 10 mmHg to 90 mmHg CO₂, 20 mmHg to 90mmHg CO₂, 30 mmHg to 90 mmHg CO₂, 40 mmHg to 90 mmHg CO₂, 50 mmHg to 90mmHg CO₂, 60 mmHg to 90 mmHg CO₂, 10 mmHg to 80 mmHg CO₂, 20 mmHg to 80mmHg CO₂, 30 mmHg to 80 mmHg CO₂, 40 mmHg to 80 mmHg CO₂, 50 mmHg to 80mmHg CO₂, 60 mmHg to 80 mmHg CO₂, 10 mmHg to 70 mmHg CO₂, 20 mmHg to 70mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHg to 70mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHg to 70mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHg to 70mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHg to 60mmHg CO₂, 30 mmHg to 60 mmHg CO₂, 40 mmHg to 60 mmHg CO₂ and 50 mmHg to60 mmHg CO₂. In various embodiments, the predetermined levels of CO₂ areadministered so that the arterial level of CO₂ reaches the PaCO₂ of anyone or more of the above ranges.

In a further embodiment, administering carbon dioxide in a stepwisemanner includes administering carbon dioxide in 30 mmHg increments inthe range of any one or more of 10 mmHg to 100 mmHg CO₂, 20 mmHg to 100mmHg CO₂, 30 mmHg to 100 mmHg CO₂, 40 mmHg to 100 mmHg CO₂, 50 mmHg to100 mmHg CO₂, 60 mmHg to 100 mmHg CO₂, 10 mmHg to 90 mmHg CO₂, 20 mmHgto 90 mmHg CO₂, 30 mmHg to 90 mmHg CO₂, 40 mmHg to 90 mmHg CO₂, 50 mmHgto 90 mmHg CO₂, 60 mmHg to 90 mmHg CO₂, 10 mmHg to 80 mmHg CO₂, 20 mmHgto 80 mmHg CO₂, 30 mmHg to 80 mmHg CO₂, 40 mmHg to 80 mmHg CO₂, 50 mmHgto 80 mmHg CO₂, 60 mmHg to 80 mmHg CO₂, 10 mmHg to 70 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 60 mmHg CO₂, 30 mmHg to 60 mmHg CO₂, 40 mmHg to 60 mmHg CO₂ and 50mmHg to 60 mmHg CO₂. In various embodiments, the predetermined levels ofCO₂ are administered so that the arterial level of CO₂ reaches the PaCO₂of any one or more of the above ranges.

In a further embodiment, administering carbon dioxide in a stepwisemanner includes administering carbon dioxide in 40 mmHg increments inthe range of any one or more of 10 mmHg to 100 mmHg CO₂, 20 mmHg to 100mmHg CO₂, 30 mmHg to 100 mmHg CO₂, 40 mmHg to 100 mmHg CO₂, 50 mmHg to100 mmHg CO₂, 60 mmHg to 100 mmHg CO₂, 10 mmHg to 90 mmHg CO₂, 20 mmHgto 90 mmHg CO₂, 30 mmHg to 90 mmHg CO₂, 40 mmHg to 90 mmHg CO₂, 50 mmHgto 90 mmHg CO₂, 60 mmHg to 90 mmHg CO₂, 10 mmHg to 80 mmHg CO₂, 20 mmHgto 80 mmHg CO₂, 30 mmHg to 80 mmHg CO₂, 40 mmHg to 80 mmHg CO₂, 50 mmHgto 80 mmHg CO₂, 60 mmHg to 80 mmHg CO₂, 10 mmHg to 70 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 60 mmHg CO₂, 30 mmHg to 60 mmHg CO₂, 40 mmHg to 60 mmHg CO₂ and 50mmHg to 60 mmHg CO₂. In various embodiments, the predetermined levels ofCO₂ are administered so that the arterial level of CO₂ reaches the PaCO₂of any one or more of the above ranges.

In a further embodiment, administering carbon dioxide in a stepwisemanner includes administering carbon dioxide in 50 mmHg increments inthe range of any one or more of 10 mmHg to 100 mmHg CO₂, 20 mmHg to 100mmHg CO₂, 30 mmHg to 100 mmHg CO₂, 40 mmHg to 100 mmHg CO₂, 50 mmHg to100 mmHg CO₂, 60 mmHg to 100 mmHg CO₂, 10 mmHg to 90 mmHg CO₂, 20 mmHgto 90 mmHg CO₂, 30 mmHg to 90 mmHg CO₂, 40 mmHg to 90 mmHg CO₂, 50 mmHgto 90 mmHg CO₂, 60 mmHg to 90 mmHg CO₂, 10 mmHg to 80 mmHg CO₂, 20 mmHgto 80 mmHg CO₂, 30 mmHg to 80 mmHg CO₂, 40 mmHg to 80 mmHg CO₂, 50 mmHgto 80 mmHg CO₂, 60 mmHg to 80 mmHg CO₂, 10 mmHg to 70 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 70 mmHg CO₂, 30 mmHg to 70 mmHg CO₂, 40 mmHg to 70 mmHg CO₂, 50 mmHgto 70 mmHg CO₂, 60 mmHg to 70 mmHg CO₂, 10 mmHg to 60 mmHg CO₂, 20 mmHgto 60 mmHg CO₂, 30 mmHg to 60 mmHg CO₂, 40 mmHg to 60 mmHg CO₂ and 50mmHg to 60 mmHg CO₂. In various embodiments, the predetermined levels ofCO₂ are administered so that the arterial level of CO₂ reaches the PaCO₂of any one or more of the above ranges.

Other increments of carbon dioxide to be administered in a stepwisemanner will be readily apparent to a person having ordinary skill in theart.

In a further embodiment, a predetermined amount of CO₂ is administeredin a block manner. Block administration of carbon dioxide comprisesadministering carbon dioxide in alternating amounts over a period oftime. “In alternating amounts” of CO₂ comprises alternating between anyof 20 mmHg and 40 mmHg, 30 mmHg and 40 mmHg, 20 mmHg and 50 mmHg, 30mmHg and 50 mmHg, 40 mmHg and 50 mmHg, 20 mmHg and 60 mmHg, 30 mmHg and60 mmHg, 40 mmHg and 60 mmHg, and 50 mmHg and 60 mmHg. In variousembodiments, the predetermined levels of CO₂ are administered so thatthe arterial level of CO₂ reaches the PaCO₂ of any one or more of theabove ranges. Other amounts of carbon dioxide to be used in alternatingamounts over a period of time will be readily apparent to a personhaving ordinary skill in the art.

In one embodiment, vascular reactivity may be measured bycharacterization of Myocardial Perfusion Reserve, which is defined as aratio of Myocardial Perfusion at Stress to Myocardial Perfusion at Rest.In healthy subjects the ratio may vary from 5:1 to 6:1. The ratiodiminishes with disease. A decrease in this ratio to 2:1 from thehealthy level is considered to be clinically significant and indicativeof poor vascular reactivity.

In another embodiment, vascular reactivity may be measured viadifferential absolute perfusion, which may be obtained using imagingmethods such as first pass perfusion, SPECT/PET, CT perfusion orechocardiography in units of ml/sec/g of tissue.

In an embodiment the CO₂ gas is administered via inhalation. CO₂ may beadministered using, for example, RespirACT™ technology from ThornhillResearch. In various embodiments, CO₂ is administered for 1-2 minutes,2-4 minutes, 4-6 minutes, 6-8 minutes, 8-10 minutes, 10-12 minutes,12-14 minutes, 14-16 minutes, 16-18 minutes and/or 18-20 minutes. In oneembodiment, CO₂ is administered for 4-6 minutes. In an additionalembodiment, CO₂ is administered for an amount of time it takes for thearterial PaCO₂ (partial pressure of carbon dioxide) to reach 50-60 mmHgfrom the standard levels of 30 mmHg during CO₂-enhanced imaging.

In one embodiment, carbon dioxide used to induce hyperemia ismedical-grade carbogen which is an admixture of 95% O₂ and 5% CO₂ Invarious other embodiments, carbon dioxide used to induce hyperemia maybe an admixture of ranges including but not limited to 94% O₂ and 6%CO₂, 93% O₂ and 7% CO₂, 92% O₂ and 8% CO₂, 91% O₂ and 9% CO₂, 90% O₂ and10% CO₂, 85% O₂ and 15% CO₂, 80% O₂ and 20% CO₂, 75% O₂ and 25% CO₂and/or 70% O₂ and 30% CO₂.

In another embodiment, vascular reactivity and/or vasodilation aremonitored using any one or more of positron emission tomography (PET),single photon emission computed tomography/computed tomography (SPECT),computed tomography (CT), magnetic resonance imaging (MRI), functionalmagnetic resonance imaging (fMRI), single photon emission computedtomography/computed tomography (SPECT/CT), positron emissiontomography/computed tomography (PET/CT), ultrasound, electrocardiogram(ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO),and electron spin resonance (ESR), and/or any combination of the imagingmodalities such as (PET/MR), PET/CT, and/or SPECT/MR In an embodiment,vascular reactivity is monitored using free-breathing BOLD MRI.

Imaging techniques using carbon dioxide involve a free-breathingapproach so as to permit entry of CO₂ into the subject's system. In anembodiment, the subjects include mammalian subjects, including, human,monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat. Ina preferred embodiment, the subject is human. It should be noted thatthe terms “subject” and “patient” are used interchangeably herein.

Advantages of the Invention

The methods described herein to functionally assess the oxygen status ofthe myocardium include administering an effective amount of CO₂ to asubject in need thereof. In an embodiment, the O₂ level is held constantwhile the CO₂ level is altered so as to induce hyperemia. Applicantsherein show the vascular reactivity in subjects in response to changesin PaCO₂. The existing methods use adenosine, dipyridamole, orregadenoson which have significant side-effects, as described above. Asdescribed in the Examples below, in some embodiments CO₂ is at least aseffective as the existing methods (which use, for example, adenosine)but without the side effects. Methods described herein may provide oneor more of the following advantages.

The use of CO₂ can provide distinct advantages over the use of, forexample, adenosine. Administering CO₂ is truly non-invasive because itmerely involves inhaling physiologically sound levels of CO₂. Theinstant methods are simple, repeatable and fast and most likely have thebest chance for reproducibility. Not even breath-holding is necessaryduring acquisition of images using the methods described herein. Theinstant methods can also be highly cost-effective as no pharmacologicalstress agents are required, cardiologists may not need to be presentduring imaging and rapid imaging reduces scan times and costs. Further,in some embodiments CO₂ can produce a selected hyperemic responsedespite consumption of caffeine, which is advantageous compared to somehyperemia-inducing drugs which do not produce reliable assessments insubjects who have consumed caffeine beforehand.

Further, the improved BOLD MRI technique described above can provide anon-invasive and reliable determination of ischemic volume (noradiation, contrast-media, or adenosine) and other value-added imagingbiomarkers from the same acquisition (Ejection Fraction, WallThickening). Additionally, the subject does not experienceadenosine-related adverse side effects and thus greater patienttolerance for repeat ischemia testing is achieved. In some embodiments,there is a significant cost-savings from abandoning exogenous contrastmedia and adenosine/regadenoson. Moreover, the proposed BOLD MRIparadigm can be accompanied by significant technical advances as well:(i) a fast, high-resolution, free-breathing 4D SSFP MRI at 3T, that canimpact cardiac MRI in general; (ii) Repeated stimulations of the heartvia precisely targeted changes in PaCO_(2;) and (iii) adoption ofsophisticated analytical methods employed in the brain to the heart.

EXAMPLES

In Examples 1-6, all imaging studies were performed in instrumentedanimals with a Doppler flow probe attached to the LAD coronary arteriesfor measurement of flow changes in response to CO₂ and clinical dose ofadenosine. In these studies, CO₂ and O₂ delivery were tightly controlledusing Respiract. CO₂ values were incremented in steps of 10 mmHgstarting from 30 mmHg to 60 mmHg and were ramped down in decrements of10 mmHg. At each CO₂ level, free-breathing and cardiac gatedblood-oxygen-level-dependent (BOLD) acquisitions were prescribed at middiastole and Doppler flow velocities were measured. Similar acquisitionswere also performed with block sequences of CO₂ levels; that is, CO₂levels were alternated between 40 and 50 mmHg and BOLD images (andcorresponding Doppler flow velocities) were acquired at each CO₂ levelto assess the reproducibility of the signal changes associated withdifferent CO₂ levels. Each delivery of CO₂ using Respiract was made inconjunction with arterial blood draw to determine the arterial blood CO₂levels. Imaging-based demonstration of myocardial hyperemic response tochanges in PaCO₂ was shown in health human volunteers with informedconsent.

Example 1

We show that CO₂ can increase myocardial perfusion by a similar amount,as does adenosine in canine models. We also show that in the setting ofcoronary artery narrowing, it is possible to detect regional variationsin hyperemic response with the use of MRI by detecting signal changes inthe myocardium due to changes in oxygen saturation (also known as theBOLD effect) using a free-breathing BOLD MRI approach.

As show in FIG. 1, a 20% BOLD signal increase (hyperemic response) withmedical-grade carbogen breathing in the absence of stenosis in dogs wasobserved. With a severe stenosis, the hyperemic response wassignificantly reduced in the LAD (left anterior descending) territorybut the other regions showed an increase in signal intensity (asobserved with adenosine). First-pass perfusion images acquired withadenosine under severe stenosis (in the same slice position and triggertime) is also shown for comparison. Heart rate increase of around 5-10%and a drop in blood pressure (measured invasively) by about 5% was alsoobserved in this animal under carbogen. All acquisitions were navigatorgated T2-prep 2D SSFP (steady-state free precession) and triggered atmid/end diastole (acquisition window of 50 ms). 10 dogs have beenstudied with medical-grade carbogen and have yielded highly reproducibleresults.

In detail, the color images (lower panel of FIG. 1) are color-coded tothe signal intensities of grayscale images (above). The darker colors(blue/black) represent territories of baseline myocardial oxygenationand the brighter regions represent those regions that are hyperemic. Onaverage the signal difference between a dark blue (low signal) andorange color (high signal) is about 20%. Note that in the absence ofstenosis, as one goes from 100% O₂ to Carbogen, the BOLD signalintensity was elevated (second image from left) suggesting CO₂ basedvasoreactivity of the myocardium. The dogs were instrumented with anoccluder over the left-anterior descending (LAD) coronary artery. As theLAD is occluded, note that the region indicated by an arrow (i.e.approximately between 11 o'clock and 1-2 o'clock (region supplied by theLAD)) becomes darker (3rd image from left), suggesting that vasodilationwas no longer possible or was reduced. The first pass image (obtainedwith adenosine stress following BOLD images) at the same stenosis levelalso shows this territory clearly. We have also compared the epicardialflow enhancements in response to Carbogen (with ETCO2 reaching 48-50 mmHg) against clinical dose of adenosine and the responses have been quitesimilar (−20% response).

Example 2

Co-Relation between Inhaled CO₂ and Oxygen Saturation

We assessed the difference between myocardial blood-oxygen-leveldependent (BOLD) response under hypercarbia and normocarbia conditionsin canines. The BOLD signal intensity is proportional to oxygensaturation.

Top panels of FIG. 2 depict the myocardial response under hypercarbia(60 mm Hg) and normocarbia (30 mmHg) conditions and show an increase inBOLD signal intensity under hypercarbia condition. The lower paneldepicts the difference as obtained by subtracting the signal under restfrom that under stress. The myocardial BOLD signal difference betweenthe two is depicted in grey and shows the responsiveness of canines tohypercarbia conditions.

We further assessed the myocardial BOLD response to stepwise CO₂increase (ramp-up) in canines. As shown in FIG. 3, as the amount of CO₂administered increases, the BOLD signal intensity increases which isindicative of an increase in hyperemic response to increased uptake ofCO₂ and oxygen saturation.

To further evaluate vascular reactivity and coronary response to CO₂, wemeasured the myocardial BOLD signal in response to block increases ofCO₂ in canines. Specifically, the myocardial BOLD signal was measured asthe amount of CO₂ administered to the canine subjects alternated between40 mmHg CO₂ and 50 mmHg CO₂. As shown in FIG. 4, an increase in CO₂level from 40 mmHg CO₂ to 50 mmHg CO₂ resulted in an increase in BOLDsignal intensity and the subsequent decrease in CO₂ level to 40 mmHgresulted in a decreased BOLD signal. These results show a tightco-relation between administration of CO₂ and vascular reactivity andcoronary response.

Example 3

Co-Relation Between the Amount of CO₂ Inhaled and Doppler Flow

Doppler flow, an ultrasound-based approach which uses sound waves tomeasure blood flow, was used to show that administration of CO₂ leads tovasodilation which results in greater blood flow, while PaO₂ is heldconstant. The Doppler flow was measured in the left anterior descending(LAD) artery. As shown in FIG. 5, as the amount of administered CO₂increases the Doppler flow increases. The relative change in coronaryflow velocity was directly proportional to the amount of CO₂administered.

Example 4

Each of the Arteries which Supply Blood to the Myocardium Responds tothe CO₂ Levels

The myocardium is supplied with blood by the left anterior descending(LAD) artery, the right coronary artery (RCA) and the left circumflex(LCX) artery. We measured the blood flow through each of these arteriesin response to increasing CO₂ supply. As shown in

FIG. 6 and summarized in FIG. 7, in each of the three LAD, RCA and LCXarteries, there is a direct correlation between the amount of CO₂administered and the signal intensity; as the amount of administered CO₂increases, the signal intensity, signaling the blood flow, in each ofthe three arteries increases. Further, as shown in FIG. 6 and summarizedin FIG. 8, there was no response to CO₂ modulation in controlterritories such as blood, skeletal muscle or air. As shown in FIG. 9,the mean hyperemic response was approximately 16%.

Example 5

Vascular Reactivity to CO₂ Comparable to Adenosine

Vascular reactivity of three canines that were administered withadenosine was compared with the vascular reactivity of canines that wereadministered with CO₂. As shown in FIG. 10, the hyperemic adenosinestress BOLD response was approximately 12% compared with 16% in responseto CO₂.

Further, as shown in FIG. 11, early human data shows a hyperemicresponse of approximately 11% for a partial pressure CO2 (pCO2) changeof l0mmHg, from 35 mmHg to 45 mmHg.

Example 6

To derive a theoretical understanding of how repeated measurements mayaffect the BOLD signal response, for a given BOLD response to PaCO₂,Applicants performed numerical simulations of statistical fits assumingvarious peak hyperemic BOLD responses to two different PaCO₂ levels (asin FIG. 12a ) along with known variability in BOLD signals. The results(FIG. 12b ) showed that as the BOLD response decreases, the number ofmeasurements required to establish statistical significance (p<0.05)associated with the BOLD response increases exponentially. This modelprovides the basis for developing a statistical framework foridentifying ischemic volume on the basis of repeated measures.

Example 7

We investigated whether a physiologically tolerable hypercapnic stimulus(˜25-mmHg increase in PaCO₂) can increase myocardial blood flow (MBF) tothat observed with adenosine in three groups of canines: (i) withoutcoronary stenosis; (ii) subjected to non-flow limiting coronarystenosis; and (iii) following pre-administration of caffeine. Thesestudies were conducted by prospectively and independently controllingPaCO₂ and combining it with ¹³N-ammonia Positron Emission Tomography(PET) measurements, and the extent of effect on MBF due to hypercapniawas compared to adenosine.

The objectives of these studies were twofold: to investigate the effectsof PaCO2 on MBF while minimizing contributions from factors that canunintentionally reduce or inaccurately report on sensitivity of PaCO2 onMBF; and to assess whether an independent, precise and rapidestablishment of physiologically tolerable level of hypercapnia providesequivalent hyperemia as adenosine, a commonly used pharmacologicalstimulus for cardiac stress testing with and without pre-administrationof caffeine. To address these aims, a clinically relevant animal modelwas used along with validated strategies for (i) precisely and rapidlyestablishing desired levels of PaCO2, while holding PaO₂ constant; (ii)quantifying MBF in vivo; and (iii) image analysis to derive MBF valuesacross the different coronary supply territories. We compared ourfindings to the effects of standard dose of adenosine in the same animalmodels with and without coronary stenosis to quantify MBF and flowdeficit regions under peak tolerable PaCO2. To determine whether MBFresponse to PaCO2 overlaps the same mechanistic path as adenosine, wequantified MBF under hypercapnia and adenosine following caffeineadministration. Studies were conducted as described in Yang, H-J et al.,J. Nucl. Med. 58: 953-960 (2017), which is hereby incorporated byreference in its entirety.

Prospectively Targeted Hypercapnia as Potent Stimulator of MBF and ItsUse for Identifying Regional Impairments in MBF and MPR

We found that prospectively targeted hypercapnia was a potent stimulatorof MBF. Data are given in FIGS. 13 and 14. FIG. 13 is a tablesummarizing the mean arterial CO₂, O₂ and hemodynamic variables ofinterest in Group Intact. FIG. 14 shows the mean global MBF and MPRresponse to hypercapnia in relation to adenosine. Global MBF underadenosine and hypercapnia were both higher than at rest (p<0.05, forboth) and were not different from one another (p=0.33). Global MBFincrease under adenosine and hypercapnia were equivalent with a marginof equivalence of 0.26 ml/min/g (⅓ of the SD of the difference in MBF,0.8 ml/min/g) at a=0.05. Global MBF normalized by rate-pressure-productunder adenosine (1.16×10⁻⁵±0.80×10⁻⁵) and hypercapnia(1.26×10⁻⁵±0.56×10⁻⁵) were significantly different from rest(0.67×10⁻⁵±0.33×10⁻⁵, p<0.05 for both), but were not different from oneanother (p=1.00). Mean global MPR with adenosine and hypercapnia weresignificantly greater than 2 and were equivalent with a margin ofequivalence of 0.50 (⅓ of the SD of the difference in MPR, 1.54) atα=0.05. The mean global MPRs did not differ under hypercapnia andadenosine (p=1.00). Similar observations were evident for regional MBFand MPR with hypercapnia and adenosine. The observed range of MBF atrest (0.44 to 1.93 ml/min/g) and adenosine (0.47 to 5.10 ml/min/g) andrange of MPR under adenosine (1.20 to 4.57) across the animals isconsistent with previous reports (Kuhle, W.G. et al., Circulation86:1004-1017 (1992)). Notably, these results indicate that an increasein MBF is not different from that are observed with adenosine and is notattributable to changes in myocardial oxygen consumption (work) indexedby rate-pressure product.

FIG. 15 shows the mean global and regional MBF and MPR response tohypercapnia in relation to adenosine. FIG. 16 shows the mean regionalMBF in LAD, left circumflex artery (LCx) and right coronary artery (RCA)supply territories at rest, hypercapnia and adenosine. MBF values werenot different at rest among the different supply territories (p>0.4, forall). MBF increased under hypercapnia and adenosine (p<0.05, for allterritories), albeit the increase in the LAD territory was significantlylower than in the LCx and RCA territories (with hypercapnia andadenosine; both p<0.05). MBF under hypercapnia was not different betweenthe LCx and RCA territories (hypercapnia: p=0.21); the same was trueunder adenosine (p=0.50). For each myocardial supply territory, MBFunder hypercapnia and adenosine were not different (p=1.00, for all).Collective comparisons of regional MBF between hypercapnia and adenosineshowed significant correlation (R=0.69, p<0.05) and good agreement(bias=0.41 ml/min/g). MPR values were not higher than 2.0 in LADterritories (hypercapnia: p=0.48 and adenosine: p=0.52) but were higherthan 2.0 in the LCx and RCA (p<0.05 for hypercapnia and adenosine)territories. MPR under hypercapnia was not different between the LCx andRCA territories (hypercapnia: p=0.59); the same was true under adenosine(p=0.34). For each myocardial supply territory, MPR under hypercapniaand adenosine were not different (p>0.5 for all). Collective comparisonsof regional MPR between hypercapnia and adenosine showed significantcorrelation (R=0.71, p<0.05) and good agreement (bias=0.21). Takentogether, FIGS. 15 and 16 show that CO₂ is able to increase blood flowand the myocardial blood flow reserve to the same extent in healthy andaffected territories of the myocardium.

Perfusion Defect Volumes and Visual Scoring Under Hypercapnia VersusAdenosine

Perfusion defect volumes and visual scoring under hypercapnia vs.adenosine were determined. The total reduction in perfusion volume (TRP,% LV) between stress and rest states is shown in FIG. 17. TRP obtainedunder hypercapnia and adenosine were not different: 25±19% (hypercapnia)vs. 27±15% (adenosine); p=0.12. Direct comparison of TRP within the samesubjects under hypercapnia and adenosine were highly correlated (R=0.85,p<0.05) and were in good agreement (bias=2%). Similar trends wereobserved from visual scoring analysis. No difference between the scoresfrom hypercapnia and adenosine was observed from a paired sampledWilcoxon signed rank test (p=0.26). Visual scores between hypercapniaand adenosine were concordant with most segments falling onto thediagonal of the scoring matrix (FIG. 18). Hypercapnia also showed highaccuracy (0.95), sensitivity (0.89) and specificity (0.98) for detectingaffected segments compared to adenosine.

The results in FIGS. 17 and 18 show that the perfusion defect identifiedwith CO₂ and adenosine are substantially the same. The perfusion defectsidentified with CO₂ or adenosine were highly correlated (R=0.85) withnegligible bias (<2%), and an increase of 25 mmHg in arterial CO₂ wassufficient to increase blood flow to the levels seen with adenosine.Further, such levels were sufficient for identifying the ischemicterritories that emerge from clinically significant coronary narrowing.Moreover, the territories that were identified to have defects usingadenosine (i.e., control positives) were accurately captured with CO₂stress as well. The corollary was also true: the regions identified withabsolute certainty to have no perfusion defectcontrol negatives) werealso identified equally accurately with CO₂. Thus, this data shows thateven visual scoring, which is commonly employed in the clinical setting,can accurately identify the presence of disease on the basis of a 25mmHg increase in arterial CO₂.

Effectr of fPreadministration of Caffeine on MBF Under HypercapniaVersus Adenosine

Next the effect of pre-administration of caffeine on myocardial bloodflow under hypercapnia vs. adenosine was determined. Mean global MBF andMPR following pre- and post-caffeine administration are shown in FIGS.19 and 20. Results showed that although there is no change in MBFbetween rest and adenosine, hypercapnia was able to induce myocardialhyperemia. There was a trend towards higher resting MBF prior tocaffeine administration but this was not statistically significant(p=0.09). However, the resting MBF normalized by rate-pressure-productwas significantly higher prior to caffeine (1.5×10⁻⁵(pre-administration) vs 1.0×10⁻⁵ (post-administration), p=0.03). Theseobservations are consistent with reports in humans (Bottcher, M. et al.,J. Nucl. Med. 36:2016-2021 (1995)) and are likely related to theinfluence of caffeine on calcium cycling at rest, which is known topromote vascular smooth muscle contraction. Mean global MBF at rest(post caffeine) and under adenosine were not different (p=1.00) and weresignificantly lower than hypercapnia (p<0.05, for both). Under caffeine,there was no correlation between MBF under adenosine and hypercapnia(R=0.02, p=0.59). Under caffeine, the global MPR under adenosine waslower than under hypercapnia (p<0.05). Regional MPR regressed againstadenosine and hypercapnia showed a weak and non-significant correlation(R=0.13, p=0.10). These findings of differential MBF response tohypercapnia and adenosine following pre-administration of caffeinesuggest that the mechanism of action mediating myocardial hyperemia bythese stimuli are at least partly different. These data also show thatcarbon dioxide can produce the requisite hyperemic response forassessing ischemic territories despite consumption of caffeine, incontrast to some hyperemia-inducing drugs which do not produce reliableassessments in subjects who have consumed caffeine beforehand.

In sum, these results show that in the absence of stenosis, mean MBFunder hypercapnia was 2.1±0.9 ml/min/g and adenosine was 2.2±1.1ml/min/g were significantly higher than at rest (0.9±0.5 ml/min/g,P<0.05); and were not different from each other (P=0.30). Underleft-anterior descending coronary (LAD) stenosis, MBF increased inresponse to hypercapnia and adenosine (p<0.05, all territories) but theeffect was significantly lower than in the LAD territory (withhypercapnia and adenosine; both p<0.05). Mean perfusion defect volumesmeasured with adenosine and hypercapnia were significantly correlated(R=0.85) and were not different (p=0.12). Following pre-administrationof caffeine, a known inhibitor of adenosine, resting MBF decreased andhypercapnia increased MBF but not adenosine (p<0.05). The resultsindicate that arterial blood CO₂ tension when increased by 25 mmHg caninduce MBF to the same level as a standard dose of adenosine.

The results thus demonstrate substantial similarity in physiologicoutcomes on several scores between a clinically-relevanthyperemia-inducing drug (adenosine) and carbon dioxide. It is notedthat, in addition to having significant negative side effects,adenosine's negative effects can last for hours whereas carbon dioxidecan be exhaled off in a few breaths. Importantly, it is not only thehyperemia inducing effect on the heart as a whole that is comparable forcarbon dioxide and adenosine; the identified blood flow deficit is alsothe same (for both location and relative volume of deficit, whichinfluences treatment choices in the clinic).

There was also excellent concordance in how blind observers (blind towhether adenosine or CO₂ was used to cause the effect) scored the imagesin terms of deficit severity. Thus, CO₂ is able to discriminate ischemicheart disease in a clinically relevant setting, indicating thatprospectively targeted arterial CO₂ can be used as an alternative tocurrent pharmacological vasodilators for cardiac stress testing.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorsthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the invention to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe invention and its practical application and to enable others skilledin the art to utilize the invention in various embodiments and withvarious modifications as are suited to the particular use contemplated.Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects. It willbe understood by those within the art that, in general, terms usedherein are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.).

What is claimed is:
 1. A method of inducing hyperemia to diagnosecoronary heart disease in a subject in need thereof, comprising:administering a 0O₂-containing gas to the subject; attaining at leastone increase in the subject's coronary PaCO₂ sufficient for diagnosingcoronary heart disease from imaging data; and imaging the heart during aperiod in which the at least one increase in PaCO₂ is measurable, toproduce imaging data indicative of a cardiovascular-disease-associatedvasoreactive response in at least one coronary blood vessel or region ofthe heart.
 2. (canceled)
 3. The method of claim 1 comprising attainingthe at least one increase in PaCO₂ in a block manner.
 4. The method ofclaim 1, comprising administering CO₂ via inhalation to attain apredetermined PaCO₂. 5.-6. (canceled)
 7. The method of claim 1, whereinthe cardiovascular disease-associated vasoreactive response is acompromised increase in blood flow.
 8. The method of claim 1, whereinthe imaging method is PET or SPECT and the measure of thecardiovascular-disease-associated vasoreactive response is the presenceor absence of a threshold increase in blood flow.
 9. The method of claim1, wherein the imaging data is indicative of the presence or absence ofa two-fold increase in blood flow.
 10. The method of claim 1, whereinthe PaCO₂ is increased and decreased in a block manner repeatedly. 11.The method of claim 1, wherein the imaging data are obtained by MRI. 12.The method of claim 1, wherein the imaging data are a change in signalintensity of a BOLD MRI signal. 13.-17. (canceled)
 18. The method ofclaim 11, further comprising: (i) registering and segmenting MRI imagesto obtain the myocardial dynamic volume, and (ii) identifying ischemicterritory and quantifying image volume.
 19. The method of claim 12,further comprising: (i) imaging the myocardium to obtain free-breathingcardiac phase resolved 3D myocardial BOLD images; (ii) registering andsegmenting the images to obtain the myocardial dynamic volume; and (iii)identifying ischemic territory and quantifying image volume. 20.-22.(canceled)
 23. A method for imaging hyperemia in a subject in need of adiagnostic assessment of cardiovascular disease comprising administeringa CO₂ containing gas in a non-therapeutic diagnostic setting, attainingat least one selected increase in a subject's coronary PaCO₂ sufficientfor diagnosing coronary heart disease from imaging data and imaging theheart during a period in which the selected increase in PaCO₂ ismeasurable, wherein the imaging data is selected to be indicative of acardiovascular-disease-associated vasoreactive response in at least onecoronary blood vessel or region of the heart. 24.-25. (canceled)
 26. Themethod of claim 23, wherein the cardiovascular-disease associatedvasoreactive response is comparable to a vasodilatory response producedby administering a hyperemia inducing drug for a duration and in amountper unit of time effective to assess coronary disease. 27.-35.(canceled)
 36. The method of claim 1, wherein the administration of theCO₂-containing gas increases the PaCO₂ in the subject by about 22 toabout 28 mm Hg.
 37. The method of claim 36, wherein the administrationof the CO₂-containing gas increases the PaCO₂ in the subject by about 25mm Hg. 38.-39. (canceled)
 40. A method for controlling a gas flowcontroller during a cardiac imaging procedure, the gas flow controlleroperable to deliver controlled amounts of carbon dioxide for inspirationby a subject during free breathing, the method comprising operating thegas mass flow controller to administer controlled amounts of carbondioxide to attain an at least one altered level of carbon dioxide in thesubject's arterial blood, the at least one altered level of carbondioxide selected to induce a selected hyperemic response in thesubject's myocardium over a time period selected for imaging theselected hyperemic response, the selected hyperemic responsepredetermined to enable at least one segment of the subject's myocardiumwith a reduced hyperemic response to be identified in the cardiacimaging procedure.
 41. The method of claim 40, wherein the reducedhyperemic response is a decrease in a ratio of myocardial perfusion atstress to myocardial perfusion at rest of 2:1. 42.-51. (canceled) 52.The method of claim 40, wherein the selected hyperemic response isinduced by attaining a carbon dioxide level of 60 to 65 mm of Hg for atleast one to two minutes.
 53. (canceled)
 54. The method of claim 40,wherein the altered level of carbon dioxide is a 25 mm of Hg increasefrom a previous level, optionally a measured baseline level for thesubject, optionally a baseline level for the subject at rest, optionallya baseline level for the subject when the subject is breathing at aregulated elevated minute volume. 55.-74. (canceled)